This invention relates to human interferon-xcex1 hybrids and nucleic acid molecules that encode these hybrids.
Interferons arc cytokines produced by a variety of eukaryotic cells upon exposure to certain environmental stimuli, including mitogens, endotoxins, double stranded RNA, and viral infection. In addition to having antiviral properties, interferons have been shown to affect a wide variety of cellular functions. These effects include inhibition of cell proliferation, immune regulatory functions and activation of multiple cellular genes. Interferons (IFNs) have been classified into four groups according to their chemical, immunological, and biological characteristics: xcex1 (leukocyte), xcex2 (fibroblast), xcex3, and xcfx89. IFNs are further identified by the eukaryote in which they originated, with HuIFN indicating human interferon, for instance.
HuIFN-xcex1s are encoded by a multigene family consisting of about 20 genes; each gene encodes a single subtype of the HuIFN-xcex1. Amino acid sequence identity among IFN-xcex1 subtypes is generally 80-85% (Horisberger and Di Marco 1995). HuIFN-xcex1 polypeptides are produced by a number of human cell lines and human leukocyte cells after exposure to viruses or double-stranded RNA, or in transformed leukocyte cell lines (e.g., lymphoblastoid lines).
IFN-xcex1s act through interaction with cell-surface receptors and induce the expression, primarily at the transcriptional level, of a broad but specific set of cellular genes. Several IFN-induced gene products have been used as markers for the biological activity of interferons. These include, for instance, ISG15, ISG54, IRF1, GBP, and IP10.
Individual IFN-xcex1 subtypes have different biological activities. For instance, it was recognized early in interferon research that IFN-xcex11 and IFN-xcex12 have distinct target-cell specificities. Human IFN-xcex12 shows high specific activity on bovine and human cells (similar to most HuIFN-xcex1s), whereas human IFN-xcex11 shows high activity only on bovine cells.
Interferon activities were first characterized in relation to viral infections, and IFN-xcex1s have proven to be remarkably effective antiviral agents. The current definition of IFN activity units is expressed in virological terms. There are many assays known to those skilled in the art that measure the degree of resistance of cells to viruses (McNeill, 1981). These assays generally can be categorized into three types: inhibition of cytopathic effect; virus plaque formation; and reduction of virus yield. Viral cytopathic effect assays measure the degree of protection induced in cell cultures pretreated with IFN and subsequently infected with viruses. Vesicular stomatitis virus, for instance, is an appropriate virus for use in such an assay. This type of assay is convenient for screening numerous different IFNs, as it can be performed in 96-well plates (Rubinstein et al., 1981). Plaque-reduction assays measure the resistance of IFN-treated cell cultures to a plaque-forming virus (for instance, measles virus). One benefit to this assay is that it allows precise measurement of a 50% reduction in plaque formation. Finally, virus yield assays measure the amount of virus released from cells during, for instance, a single growth cycle. Such assays are useful for testing the antiviral activity of IFNs against viruses that do not cause cytopathic effects, or that do not build plaques in target-cell cultures. The multiplicity of infection (moi) is an important factor to consider when using either plaque-reduction or virus-yield assays.
Other clinically important interferon characteristics are also easily assayed in the laboratory setting. One such characteristic is the ability of an interferon polypeptide to bind to specific cell-surface receptors. For instance, some IFN-xcex1s exhibit different cell-surface properties compared to IFN-xcex12b, the IFN most widely used in clinical trials. While IFN-xcex12b is an effective antiviral agent, it causes significant adverse side effects. Interferons that exhibit distinct binding properties from IFN-xcex12b may not cause the same adverse effects. Therefore, interferons that compete poorly with IFN-xcex12b for binding sites on cells are of clinical interest. Competitive interferon binding assays are well known in the art (Hu et al., 1993; Di Marco et al., 1994). In general, such assays involve incubation of cell culture cells with a mixture of 125I-labeled IFN-xcex12b and an unlabeled interferon of interest. Unbound interferon is then removed, and the amount of bound label (and by extension, bound 125I-labeled IFN-xcex12b) is measured. By comparing the amount of label that binds to cells in the presence or absence of competing interferons, relative binding affinities can be calculated.
Another prominent effect of IFN-xcex1s is their ability to inhibit cell growth, which is of major importance in determining anti-tumor action. Growth inhibition assays are well established, and usually depend on cell counts or uptake of tritiated thymidine ([3H]thymidine) or another radiolabel. The human lymphoblastoid Daudi cell line has proven to be extremely sensitive to IFN-xcex1s, and it has been used to measure antiproliferative activity in many IFN-xcex1s and derived hybrid polypeptides (Meister et al., 1986). Use of this cell line has been facilitated by its ability to be grown in suspension cultures (Evinger and Pestka, 1981).
IFN-xcex1s also exhibit many immunomodulatory activities (Zoon et al., 1986).
Although IFNs were first discovered by virologists, their first clinical use (in 1979) was as therapeutic agents for myeloma (Joshua et al., 1997). IFN-xcex1s have since been shown to be efficacious against a myriad of diseases of viral, malignant, angiogenic, allergic, inflammatory, and fibrotic origin (Tilg, 1997). For instance, IFN-xcex1 is the only drug that is currently approved for treatment of hepatitis C in Europe and North America (Moussalli et al., 1998), and is the treatment of choice for chronic acute hepatitis B and AIDS-related Karposi""s sarcoma. It has also proven efficacious in the treatment of metastatic renal carcinoma and chronic myeloid leukemia (Williams and Linch, 1997). Clinical uses of IFNs are reviewed in Gresser (1997) and Pfeffer (1997).
Standard recombinant techniques have become useful methods for the production and modification of IFN-xcex1 proteins (Streuli et al., 1981; Horisberger and Di Marco 1995; Rehberg et al., 1982; Meister et al., 1986; Fidler et al., 1987; Sperber et al., 1993; Mitsui et al., 1993; Muller et al., 1994; and Zav""Yalov and Zav""Yalov 1997). One such recombinant modification is the formation of hybrid IFN molecules. Hybrid IFNs contain fragments of two or more different interferon polypeptides, functionally fused together. The first IFN-xcex1 hybrids were designed to study molecular structure-function relationships. Much research has since been directed toward the production of hybrid IFNs that combine different biological properties of the parental proteins. Some hybrid IFNs display biological activity that is significantly different from that of both parent molecules (Horisberger and Di Marco 1995). For instance, certain early IFN-xcex1/IFN-xcex1 hybrids acquired the novel property of very high activity on mouse cells (Streuli et al., 1980; Rehberg et al., 1982).
The techniques used by researchers to generate hybrid IFN polypeptides have evolved through time (Horisberger and Di Marco 1995). Early researchers took advantage of the presence of naturally occurring restriction endonuclease (RE) cleavage sites within IFN-encoding sequences to piece together homologous coding fragments. (See, for instance, U.S. Pat. No. 5,071,761 xe2x80x9cHybrid Interferonsxe2x80x9d). Though convenient, this was a limited method in that only so many of such pre-existing RE sites occurred in each IFN coding sequence. In addition, the location of each restriction site was fixed, making the possible combinations relatively small. More recently, researchers have used PCR amplification to create specific desired nucleic acid fragments, thereby gaining the ability to piece together new pieces of different IFNs (Horton et al., 1989).
A number of U.S. patents discuss various hybrid IFNs, how to produce them, and how to use them to treat patients. Many such patents relate to inter-group (multi-class) hybrid IFNs, wherein portions of the final hybrid are taken from at least two different interferon classification groups (e.g., xcex1 and xcex2). For instance, U.S. Pat. No. 4,758,428 (xe2x80x9cMulticlass hybrid interferonsxe2x80x9d) describes the multi-class hybrid IFN HuIFN-xcex11(1-73)/HuIFN-xcex21(74-166), and its use in pharmaceutical compositions to treat viral infections and tumorous growths in animal patients. Another such patent (U.S. Pat. No. 4,914,033 xe2x80x9cStructure and properties of modified interferonsxe2x80x9d) discloses the making of constructs that encode hybrid interferons comprising amino- and carboxy-terminal fragments of HuIFN-xcex2 fused to an internal sequence (amino acid residues 36-46) of a HuIFN-xcex1. This patent also discloses the purification of the encoded hybrid IFN polypeptide and its use in pharmaceutical formulations.
Intra-group hybrid interferons (e.g., xcex11/xcex18 hybrids) have also been described. U.S. Pat. No. 5,071,761 (xe2x80x9cHybrid interferonsxe2x80x9d) provides a good example of such intra-group hybrids. This patent discloses the construction, purification, use, and pharmaceutical preparation of various fusions hybrids between HuIFN-xcex11 and HuIFN-xcex18, where as many as four distinct IFN-xcex1 fragments have been used to construct the fusion. The construction, purification, and use of similar IFN-xcex1 hybrids to treat animal patients are disclosed in U.S. Pat. No. 5,137,720 (xe2x80x9cAntiviral combination, and method of treatmentxe2x80x9d).
It is to such engineered, recombinant intra-group hybrid interferon molecules that the present invention is directed.
The present invention provides hybrid interferons constructed by combining portions of two or more interferon-xcex1s, and mutant and mutant hybrid interferons constructed by point mutagenesis. These interferon molecules have good antiviral and antiproliferative activities. Thus, they may be used clinically to treat viral infections (such as influenza, rabies, and hepatitis B) and tumors, including but not limited to osteogenic sarcoma, multiple myeloma, nodular, poorly differentiated lymphoma, leukemia, carcinoma, melanoma, and papilloma, as well to modulate the immune system.
Six of the hybrids provided by this invention are termed HY-1, HY-2, HY-3, HY-4, HY-5, and HY-6, and are composed as follows:
HY-1: IFN-xcex121a(1-75)/IFN-xcex12c(76-166);
HY-2: IFN-xcex121a(1-95)/IFN-xcex12c(96-166);
HY-3: IFN-xcex12c(1-95)/IFN-xcex121a(96-166);
HY-4: IFN-xcex1-21a(1-75)/IFN-xcex12c(76-81)/IFN-xcex121a(82-95)/IFN-xcex12c(96-166);
HY-5: IFN-xcex1-21a(1-75)/IFN-xcex121a(76-81)/IFN-xcex12c(82-95)/IFN-xcex12c(96-166);
HY-6: IFN-xcex121a(1-75)/IFN-xcex12c(76-95)/IFN-xcex121a(96-166).
This nomenclature indicates that HY-1 is comprised of amino acids 1-75 of IFN-xcex121a fused to amino acids 76-166 of IFN-xcex12c; HY-2 is comprised of amino acids 1-95 of IFN-xcex121a fused to amino acids 96-166 of IFN-xcex12c; HY-3 is comprised of amino acids 1-95 of IFN-xcex12c fused to amino acids 96-166 of IFN-xcex121a; and so forth for the remaining mutants. HY-3 is 165 amino acids long due to facilitated alignment numbering, as explained below.
Further aspects of the invention include the hybrid IFNs HY-1, HY-2, HY-3, HY-4, HY-5, and HY-6 and nucleic acid molecules that encode these hybrid interferons. Also encompassed within the scope of the invention are recombinant vectors that comprise such a nucleic acid molecule. Such vectors can be transformed into various cells to gain expression of these hybrid interferons. Accordingly, the invention also encompasses a cell transformed with a recombinant vector comprising such a nucleic acid molecule.
While each of these hybrids has particular advantages for clinical use, HY-3 in particular shows striking antiproliferative activity. This activity is associated with the combination of the 76-95 region of IFN-xcex12c and the 96-166 region of IFN-xcex121a. Accordingly, another aspect of the invention comprises HY-3-like molecules comprising such IFN components. Such molecules may be represented as X-A-B wherein xe2x80x9cXxe2x80x9d comprises about amino acid residues 1-75 of any IFN-xcex1, xe2x80x9cAxe2x80x9d comprises about amino acid residues 76-95 of IFN-xcex12c and xe2x80x9cBxe2x80x9d comprises about amino acid residues 96-166 of IFN-xcex121a. While precise numerical limitations for the size of these sub-regions are provided (e.g., about amino acid residues 76-95), one of ordinary skill in the art will appreciate that these biological molecules may be varied in exact size. In certain embodiments, such variations will be no greater than plus or minus five amino acids from the specified termination points. Likewise, residues 81-90 or 81-95 of IFN-xcex12c comprise about the same amino acid residues as residues 76-95.
Strong antiproliferative activity may also be obtained by combining about IFN-xcex12c(76-95) with amino- and carboxy-regions of other IFNs. In such hybrids, the aminoxe2x80x94(about residues 1-75) and carboxyxe2x80x94(about residues 96-166) regions may be provided from a single IFN-xcex1, or from two different IFN-xcex1s. These hybrid IFN molecules may be represented as X-A-Y, wherein xe2x80x9cXxe2x80x9d comprises about amino acid residues 1-75 of any IFN-xcex1, xe2x80x9cAxe2x80x9d comprises about amino acid residues 76-95 of IFN-xcex12c, and xe2x80x9cYxe2x80x9d comprises about amino acid residues 96-166 of any IFN-xcex1. Another aspect of the invention is a recombinant IFN hybrid protein comprising first, second, and third domains, wherein the first domain comprises the amino-region of an IFN-xcex1, the second domain comprises the middle region of IFN-xcex12c (about residues 76-95), and the third domain comprises the carboxy-region of an IFN-xcex1.
In certain embodiments of the invention, a shorter region of IFN-xcex12c contained within the region from residue 76 to residue 95 will be sufficient to confer substantial antiproliferative activity on a hybrid interferon containing such a fragment. The amino- and carboxy-terminal regions are provided from a single IFN-xcex1 or from two different IFNxe2x80x2xcex1s. Such a hybrid interferon-xcex1 molecule with a short IFN-xcex12c middle region may be represented as V-C-Y, wherein xe2x80x9cVxe2x80x9d comprises about amino acid residues 1-81 of an interferon-xcex1, xe2x80x9cCxe2x80x9d comprises about amino acid residues 82-95 of IFN-xcex12c, and xe2x80x9cYxe2x80x9d comprises about amino acid residues 96-166 of an interferon-xcex1.
In particular embodiments of the invention, the third domain of the protein comprises about amino acid residues 96-166 of IFN-xcex121a. In these embodiments, the first domain of the protein comprises the amino-region of any IFN-xcex1. Such a hybrid IFN can be represented generally as X-A-B, wherein xe2x80x9cXxe2x80x9d comprises about amino acid residues 1-75 of an interferon-xcex1, xe2x80x9cAxe2x80x9d comprises about amino acid residues 76-95 of IFN-xcex12c, and xe2x80x9cBxe2x80x9d comprises about amino acid residues 96-166 of IFN-xcex121a.
Hybrid interferon molecules according to the present invention can also contain more than three segments or domains of different parental interferons. Such multiple domains are taken from at least two different source or parental interferons, and may be taken from up to as many different interferon-xcex1s as there are segments assembled to construct the hybrid. For instance, a four-domain hybrid interferon-xcex1 will be constructed from as few as two or as many as four different interferon-xcex1s.
One four domain hybrid interferon-xcex1 molecule encompassed within the current invention can be designated M-N-O-P, wherein xe2x80x9cMxe2x80x9d comprises about amino acid residues 1-75 of interferon xcex121a, xe2x80x9cNxe2x80x9d comprises about amino acid residues 76 to 81 of interferon-xcex12c, xe2x80x9cOxe2x80x9d comprises about amino acid residues 82 to 95 of interferon-xcex121a, and xe2x80x9cPxe2x80x9d comprises about amino acid residues 96 to 166 of interferon-xcex12c. A representative four domain hybrid interferon-xcex1 of this type is HY-4.
If a parental interferon that has one or more point or short deletions (as found with the 44th position in IFN-xcex12c) is used in construction of any of the hybrid interferons disclosed herein (e.g., those represented generally as X-A-B, X-A-Y, V-C-Y, or M-N-O-P), the numbering of the resultant hybrid fusions should be carried out using the facilitated alignment system.
The invention also provides nucleic acid molecules that encode any of the multi-domain hybrid IFN proteins disclosed herein, including those that can be represented generally as X-A-B, X-A-Y, V-C-Y, and M-N-O-P, as well as recombinant vectors that comprise such a nucleic acid molecule and cells transformed with such a vector.
One of ordinary skill in the art will also appreciate that minor modifications to the IFN-xcex1 sequences described herein may also be employed, such as amino acid substitutions, additions, and deletions, to create a mutant hybrid interferon-xcex1. Thus, it is entirely possible that hybrid IFNs having greater than or fewer than 166 amino acids may be produced. Substitutions will typically be conservative in nature (e.g., one aliphatic amino acid for another), and such modifications will generally be designed not to have a significant effect on the biological properties of the hybrid IFN.
Also encompassed are purified or isolated interferon-xcex1s (such as IFN-xcex12c) that contain point mutations at either residue 86 or residue 90, thereby changing these residues to tyrosine. Such mutant interferon-xcex1s may be mutant hybrid molecules, and such mutant hybrids can contain short or long segments of IFN-xcex12c, IFN-xcex121a, or both of these parental interferons. Specific representatives of these mutant hybrid interferons include SDM-1 and SDM-2. Additional mutations can be made to replace existing tyrosine residues at 86 or 90 with other amino acids; specific representatives of this type of mutant hybrid interferon are SDM-3, and SDM-4.
Further aspects of the invention include nucleic acid molecules that encode the mutant hybrid interferons as disclosed herein, and particularly SDM-1, SDM-2, SDM-3, and SDM-4. Recombinant vectors that comprise such a nucleic acid molecule are also encompassed. Such vectors can be transformed into various cells to gain expression of these mutant interferons. Accordingly, the invention also encompasses a cell transformed with a recombinant vector comprising such a nucleic acid molecule.
The invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable vehicle or carrier and at least one hybrid IFN-xcex1 polypeptide as described above. Such hybrid IFN-xcex1s include those generally represented as X-A-Y, as X-A-B, as V-C-Y, and as M-N-O-P, as well as the specific hybrids HY-1, HY-2, HY-3, HY-4, HY-5, and HY-6. Mutant hybrid IFN-xcex1s (e.g., SDM-1, SDM-2, SDM-3, or SDM-4) may also be included in such pharmaceutical compositions, either singly, in combinations with other mutant hybrid interferons, or in combination with hybrids IFNs as listed above.
These pharmaceutical compositions can be administered to humans or other animals on whose cells they are effective, in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, and subcutaneously. Accordingly, a further aspect of the invention is such a pharmaceutical composition that is an injectable composition.
The invention also encompasses methods for treating a patient for a viral disease, comprising administering to the patient a therapeutically effective, viral disease-inhibiting amount of one or more hybrid or mutant hybrid interferon-xcex1s as described above. One specific aspect of this invention is a method of treatment, wherein the hybrid interferon-xcex1 is administered to the patient by injection.
Another aspect of the invention encompasses methods for regulating cell growth in a patient, comprising administering to the patient a therapeutically effective, cell growth-regulating amount of one or more hybrid or mutant hybrid interferon-xcex1s as described above. The cell growth regulated by this treatment may be, for instance, tumor cell growth. One specific aspect of this invention is a method of regulating cell growth, wherein the hybrid or mutant hybrid interferon-xcex1 is administered to the patient by injection.